Inhibition of PI3K/AKT molecular pathway mediated by membrane estrogen receptor GPER accounts for cryptotanshinone induced antiproliferative effect on breast cancer SKBR-3 cells

Background: Breast cancer is the most frequently diagnosed cancer among women and the second leading cause of cancer death. Especially nuclear estrogen receptor (nER) negative breast cancer is always with much poor prognosis. In recent years, it was reported that membrane G protein coupled estrogen receptor (GPER), a newly recognized estrogen receptor took essential part in the development and treatment of breast cancer, especially nER negative breast cancer. The present study aimed to investigate the anti nER negative breast cancer effect and its possible molecular pathway of cryptotanshinone (CPT), an important active compound of traditional Chinese medicine Danshen. Methods: The following in vitro tests were performed in nER negative but GPER positive breast cancer SKBR-3 cells. The effect of CPT on cell proliferation rate and cell cycle distribution was evaluated by MTT cell viability test and flow cytometry assay. The role of PI3K/AKT pathway and the mediated function of GPER were tested by western blot and immunofluorescence. Technique of gene silence and the specific GPER agonist G-1 and antagonist G-15 were employed in the experiments to further verify the function of GPER in mediating the anticancer role of CPT. Results: The results showed that proliferation of SKBR-3 cells could be blocked by CPT in a time and dose dependent manner. CPT also exerted antiproliferative activities by arresting cell cycle progression in G1 phase and down regulating the expression level of cyclin A, cyclin B, cyclin D and cyclin-dependent kinase 2 (CDK2). The antiproliferative effect of CPT was further enhanced by G1 and attenuated by G15. Results of western blot and immunofluorescence both showed that PI3K and p-AKT protein expression could be down regulated by CPT and such effects were mediated by GPER which is further demonstrated by GPER gene silence test. Conclusion: Our present study shows that the antiproliferative action of CPT on SKBR-3 cells is realized by inhibition of GPER mediated PI3K/AKT pathway. These findings deserve further validation for serving as useful therapeutic targets.